The main long term goals of this project are to understand, in a fundamental sense, the catalytic strategies of RNA enzymes, and to understand the physical and chemical principles underlying this catalysis. In addition, catalysis provides a sensitive probe of structure-function relationships of RNA, some of which may be general to other RNA molecules. Finally, this understanding may aid in the design of RNA enzymes that can specifically and efficiently cleave RNA targets in vivo. The RNA enzyme or "ribozyme" derived from the intervening sequence of Tetrahymena pre-rRNA, which catalyzes a site-specific endonuclease reaction analogous to the first step of self-splicing, has been chosen for study because it is the best characterized RNA catalyst. Thus, further investigation provides the opportunity to unravel fundamental aspects of RNA catalysis. Specific aims are as follows: 1. Directly determine the rate constant of the chemical step of the Tetrahymena ribozyme reaction using rapid quench techniques. Investigate the origin of an apparent pKa near 7. Perform additional measurements to complete the kinetic and thermodynamic framework of the ribozyme reaction. This allows isolation of individual reaction steps, which is crucial for dissecting the catalytic strategies of the ribozyme. 2. Vary in a systematical and rational fashion, the reaction substrates (the guanosine nucleophile and the oligonucleotide that is cleaved) in order to reveal the types of interactions important in binding and catalysis. Analysis of these series of substrates with mutant ribozymes (generated both by site-directed mutagenesis and in vitro selection) will reveal further aspects of these interactions and their energetics. 3. Compare the binding of bisubstrate and transition state analogs to the binding of the individual substrates and products. These comparisons and the results from the experiments of Specific Aim 2 are expected to provide insight into the catalytic strategies employed by the ribozyme.